The affinity of IgG antibodies to gag p24 and p17 in HIV-1-infected patients correlates with disease progression.

The affinity of anti-gag antibody was studied for up to 9 years (1984-1993) in sera from 15 HIV-1+ patients with haemophilia. On the basis of their 1993 clinical status patients were divided into two groups: (i) patients who remained asymptomatic (n = 9); and (ii) those who progressed to AIDS between late 1987 and 1993. The affinity constants of antibody for p24 and p17 were determined by a double isotope fluid-phase radioimmunoassay; and the relationships between antibody affinity and titre, patient clinical course, CD4 cell counts and p24 antigenaemia were analysed. The affinity of p24- and p17-specific antibody was up to 100 times greater in asymptomatic patients than in patients who progressed to AIDS. Patients who developed AIDS either lost or failed to develop high-affinity antibodies early in the infection. Asymptomatic patients maintained high-affinity antibodies for several years; however, in some of these patients the affinity of anti-p24 and p17 antibodies subsequently fell later in the study period. The presence of low-affinity antibody and progressive reduction in the titre of specific antibody were earlier predictors of disease onset than CD4 cell counts. The failure to either develop or maintain high affinity gag-specific antibody suggests an early impairment of T helper function in individuals who progressed to AIDS. The presence of antibody of high affinity could be essential in controlling virus replication and the onset of AIDS.     

Differential maturation of avidity of IgG antibodies to gp41, p24 and p17 following infection with HIV-1

We have evaluated solid-phase ELISA IgG antibody avidity studies as a means of identifying cases of recent HIV-1 infection. Although separate studies on the avidity of anti-gp41 and anti-p24 antibodies in seroconvertors have been reported, a comparison of the ability of patients to simultaneously mature their immune response to more than one HIV antigen immediately following seroconversion appears to be lacking. We have demonstrated a maturation in anti-gp41 avidity which reflects the time since seroconversion in all cases. In contrast, however, only some patients produced high-avidity anti-p24 or anti-p17 antibodies during the same time span. While the avidity of anti-gp41 antibodies remained high in cases of non-recent HIV infection, even in the face of advanced disease, we have confirmed the findings of others that the avidity of anti-p24 falls before the onset of ARC or AIDS. Therefore, whilst the avidity of anti-gp41 antibodies could reliably be of value in identifying cases of recent HIV infection, the avidity of anti-p24 or anti-p17 antibodies could not, but may be of prognostic value, even at an early stage. The time taken to reach maximum anti-p17, anti-p24 and anti-gp41 titres was variable, but anti-gp41 titres, like anti-gp41 avidity, remained high. In contrast, anti-p24 titres fell, even during the early follow-up period in some seroconvertors. Anti-p24 antibody avidity, however, appeared to be a better predictor of disease progression in ‘remote’ cases than anti-p24 titre. The avidity and titres of these antibodies are presented in relation to the clinical details, p24 antigen status, CD4 and CD8 counts where these are known.     

T cell responses to peptides covering the gag p24 region of HIV-1 occur in HIV-1 seronegative individuals.

We demonstrate that peptides (16 amino acids long) covering the sequence of the HIV-1 core protein p24 induce significant proliferation in peripheral blood mononuclear cells (PBMC) of several (greater than 50%) healthy seronegative volunteers as well as seronegative homosexual men. The nature of this response was characterized and compared with those of HIV-infected patients. Several peptides induced responses; however, the most frequent responses in both seropositive and seronegative individuals were noted to the following peptides: 1 and 2 (aa 133-157); 6 and 7 (aa 183-207); 15 (aa 273-287); and 17 and 18 (aa 293-317). The response pattern was related to the disease stage of the patients; seronegative individuals as well as asymptomatic seropositive individuals (CDC II/III) responded to low concentrations of several peptides, but symptomatic patients (CDC IV) only responded to high concentrations of a few peptides. Cell separation studies of PBMC from healthy volunteers showed that the responding cells were CD4+ and expressed the CD45RO differentiation antigen. Furthermore, cord-blood mononuclear cells with less than 5% of CD45RO T cells did not proliferative to any of the peptides. Finally, CD4+ T cell lines specific for both peptides and p24 protein were successfully established from the PBMC of seronegative individuals confirming the data obtained with freshly isolated cells. These studies therefore suggest that the CD4+ cell response to p24 is not strictly disease related, instead, the response may be due to priming of the host with cross-reactive antigens.     

HIV-1 gag基因p17+24重组抗原表达、纯化及抗原性分析

目的获得HIV-1型gag基因p17＋2，4重组抗原，分析其免疫原性和探讨开发诊断试剂的可能性。方法采用PCR技术扩增HIV-1gag基因p17＋p24核酸片段，并克隆入pTreHis2A表达质粒，在大肠杆菌BL21（DE3）株中表达。用Ni—NTA金属螯合层析法纯化目的蛋白，并用免疫印迹法分析纯化蛋白的抗原特异性。结果重组质粒在BL21（DF3）菌株中经IPm诱导表达一个相对分子质量（肘，）约为51×10^3的融合重组蛋白，重组蛋白经HIV-1p17、p24抗原单克隆抗体鉴定，具有抗原特异性。25份HIV阳性血清、180份HIV阴性血清标本初步经l临床验证，具有较高的检出敏感性和特异性。结论成功构建HIV-1型gag基因p17＋24抗原表达载体，表达纯化的p17＋24重组抗原具有较好的抗原性，可用于诊断试剂的开发。     

抗HIV-1核心抗原p24单克隆抗体的制备及特性的初步鉴定

目的 :建立分泌抗HIV 1p2 4单克隆抗体 (mAb)的杂交瘤细胞株 ,并对其特性进行初步鉴定。方法 :以纯化的基因工程制备的p2 4抗原免疫BALB/c小鼠 ,取免疫小鼠的脾细胞与Sp2 /0骨髓瘤细胞融合 ,经HAT、HT选择培养及有限稀释法进行克隆化后 ,用间接ELISA法及Dotblot对其进行筛选和特性鉴定。结果 :筛选到 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,其腹水效价为 1×10 -5,亲和力为 1.7× 10 4～ 1.8×10 4mol/L ,mAb的Ig亚类均为IgG1。两株mAb与HBcAg、HCVRNA阳性血清及HIVgp4 1等均无交叉反应 ,只与HIV 1p2 4抗原阳性血清产生特异反应。结论 :成功地建立了 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,为进一步研制HIV 1p2 4抗原的ELISA检测试剂盒奠定了基础     

Antibody response and avidity of respiratory syncytial virus-specific total IgG, IgG1, and IgG3 in young children

Respiratory syncytial virus (RSV) is a major cause of acute respiratory disease in infants and young children. Considering that several aspects of the humoral immune response to RSV infection remain unclear, this study aimed to investigate the occurrence, levels, and avidity of total IgG, IgG1, and IgG3 antibodies against RSV in serum samples from children ≤5 years old. In addition, a possible association between antibody avidity and severity of illness was examined. The occurrence and levels of RSV-specific IgG depended on age, with infants <3 months old displaying high levels of antibodies, which were probably acquired from the mother. Children ≥24 months old also showed frequent occurrence and high levels of IgG, which was produced actively during infection. In addition, the avidity assay showed that the avidity of RSV-specific total IgG and IgG1 was lower in infants <3 months old who had acute respiratory disease than in age-matched controls. The avidity of RSV-specific IgG detected in children ≥24 months old with lower respiratory infection was lower than that in children with upper respiratory infection. These results indicate that the presence of high avidity RSV-specific IgG antibodies may lead to better protection against RSV infection in children <3 months old, who may have a lower probability of developing disease of increased severity. In addition, children ≥24 months old with RSV-specific IgG antibodies of low avidity tended to develop more severe RSV illness. These findings may be helpful in establishing vaccination schedules when a vaccine becomes available.     

Preparation and characterization of three monoclonal antibodies against HIV-1 p24 capsid protein

HIV-1 p24 detection provides a means to aid the early diagnosis of HIV-1 infection, track the progression of disease and assess the efficacy of antiretroviral therapy. In the present study, three monoclonal antibodies （mAbs） p3JB9, p5F1 and p6F4 against HIV-1 p24 were generated. All mAbs could detect p24 of HIV-1ⅢB, HIV-1Ada-M, HIV-174v mAbs p5F1 and p6F4 could detect HIV-1KM018, while p3JB9 could not. Three mAbs did not react with HIV-2ROD, HIV-2CBL-20 and SIVagmTYO-1. The recognized epitope of p5F1 was located on the Gag amino acid region DCKTILKALGPAATLEEMMTAC. The p5F1 was used to establish a modified sandwich ELISA with rabbit anti-p24 serum and showed good specificity and high sensitivity, which has been used to measure HIV-1 p24 antigen levels in research. Cellular ＆ Molecular Immunology.     

Quantitative measurement of cytomegalovirus-specific IgG and IgM antibodies in relation to cytomegalovirus antigenaemia and disease activity in kidney recipients with an active cytomegalovirus infection

In a longitudinal investigation 103 kidney recipients were studied with respect to the development of cytomegalovirus (CMV) specific antibodies of the IgG and IgM class, in relation to the detection of CMV antigenaemia (immediate early antigen, IEA), in weekly obtained blood samples during the first 3 months after transplantation. In 15 out of 49 (31%) seronegative patients a primary infection occurred, which was characterized by a quick rise in IgM antibody followed by a slower production of IgG antibody, high maximum numbers of IEA+ cells, and a CMV syndrome in 11 patients. In 35 out of 54 (65%) seropositive patients a secondary infection occurred. After a post-operative fall in the IgG antibody, which was also found in patients without an active infection and which was accompanied by a similar drop in serum albumin and IgG, a second dip in IgG antibody was found 6 days before the first IEA+ leucocyte appeared in the blood. This was followed by a significant increase, indicative of an active immune response in consequence of the infection, 18 days later. In 31 of these 35 patients an IgM response was found. This could be ascribed to the presence of rheumatoid factor activity in 20 of them. Eight patients who showed a transient rise in IgG antibody between the two dips could be distinguished from the remaining ones by a lower maximum number of IEA+ cells and less severe disease symptoms. The described results suggest that (i) an adequate humoral immune system may prevent symptomatic CMV disease in secondary infections; and (ii) CMV-specific antibodies may be removed from the circulation by antigens present in infected tissues before CMV antigenaemia becomes detectable.     

The restricted IgG1 antibody response to maedi visna virus is seen following infection but not following immunization with recombinant gag protein

Maedi-visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV), infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgGi subclass-restricted MVV-neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti-Orf antibody is readily detected. By ELISA, the IgGi antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti-MVV gag p. 25 can be detected in both normal and MVV-infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freund's complete adjuvant. The failure to make an IgG2 MVV-specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence.     

The presence of antibodies to purified p24gag protein of HTLV-I in sera of patients with systemic lupus erythematosus (SLE)

Sera of patients with systemic lupus erythematosus (SLE) were tested for their reactivity to HTLV-I by western blotting (WB). Seven (18%) of 40 SLE serum samples reacted to the p24^gag protein of HTLV-I by WB using purified gag antigens. The specificity of anti-p24^gag antibodies in the SLE sera was confirmed by competitive inhibition on WB. Two of the seven patients were shown to be HTLV-I carriers, because HTLV-I infected T cell lines were easily established from their peripheral blood mononuclear cells (PBMC). Except for these two carrier patients, the gag proteins were not detected in the lysates of PBMC by WB using anti-p24^gag and anti-p19^gag monoclonal antibodies. The gag and pX genes of HTLV-I were not detected by PCR in PBMC of the SLE patients, with the exception of the 2 HTLV-I carrier patients. These results show no direct involvement of HTLV-I in the etiology of SLE. However, the existence of a specific antibody to p24^gag in the sera of some of the noncarrier SLE patients suggests a crossreactivity to either unknown viruses or some autoantigens.     

Long-lasting humoral immune response induced in HIV-1-infected patients by a synthetic peptide (AT20) derived from the HIV-1 matrix protein p17 functional epitope

Objective:

A therapeutic vaccination based on a synthetic peptide (AT20) representative of the HIV-1 matrix protein p17 (p17) functional region, coupled to keyhole limpet hemocyanin (KLH) AT20-KLH was capable of inducing the production of high-avidity antibodies (Abs) toward a previous untargeted p17 hotspot of functional activity in highly active antiretroviral therapy (HAART)-treated HIV-1-infected patients. Since avidity of Abs after immunization and the retention of antigens are important in sustaining the long-lasting production of specific humoral responses, we asked whether AT20-KLH vaccination would result in development of a long-lived immune response.

Methods:

The long-term duration of Ab response to AT20-KLH has been evaluated in 10 patients previously enrolled for the AT20-KLH vaccination trial at day 898 post-immunization. Ab titer and their avidity was assessed using specifically designed ELISA assays, whereas their neutralizing capacity was estimated in vitro using a ‘wound sealing assay’.

Results:

Data obtained show that high titers of specific anti-AT20 Abs were maintained at more than 2?years after the last immunization. Furthermore, these Abs were capable to neutralize exogenous p17, as assessed by ability of sera derived from AT20-KLH-immunized patients to block the ability of p17 to promote cell migration in vitro.

Conclusion:

This finding attests for a successful AT20-KLH vaccine molecule formulation and for an effective HAART-dependent Ab persistence.     

表达HIV-1 gag蛋白重组乳酸乳球菌株的建立及其在小鼠体内定植的研究

目的 构建表达HIV-1 gag的重组乳酸乳球菌,并分析重组菌在小鼠体内的定植情况.方法 将HIV-1 gag克隆至原核表达质粒pMG36e,重组质粒pMG36e-gag电转乳酸乳球菌,SDS-PAGE和Western blot法检测其在乳酸乳球菌中的表达.以109个重组菌给小鼠口服,对照组口服PBS.口服后第1、3、5、7、9、11天分别取小鼠的胃、小肠和大肠,采用稀释滴种法测定小鼠胃、肠道内重组菌的数量.结果 酶切和DNA测序表明gag已克隆入pMG36e.SDS-PAGE在预期位置检测到蛋白条带,Western blot分析证实其为gag蛋白.重组菌定植实验表明,第1、3天在胃肠均有一定量重组菌,且大肠中重组菌数量多于胃和小肠,随后胃肠中重组菌均迅速减少,仅在大肠中仍有一定量的重组菌,第11天时胃肠仅分离到极少量的重组菌.结论 本研究获得胞内表达HIV-1 gag前体蛋白Pr55的重组乳酸乳球菌,其可在胃肠中短期存活,可作为预防HIV-1感染的候选疫苗.     

Immune responses to six synthetic peptides of capsid protein with sera from HIV-1 infected individuals

Many B cell epitopes within p24 of human immunodeficiency virus type 1 （HIV-1） were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera （19.4%）, all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals.     

Infection of human CD4+ rabbit cells with HIV-1: the possibility of the rabbit as a model for HIV-1 infection.

Although human T cell surface glycoprotein CD4 is the cellular receptor for human immunodeficiency virus 1 (HIV-1), the introduction of the human CD4 gene into murine cells does not render them susceptible to HIV-1 infection. Here we have established rabbit transfectant cell lines expressing human CD4 on the cell surface and demonstrated that the CD4+ rabbit transfectants could be readily infected by HIV-1 by co-cultivating with a HIV-1-infected human MOLT-4 T cell line (MOLT-4/HIV). Avid syncytia formation was observed upon co-cultivation and the syncytia abundantly produced HIV-1 mature particles, as revealed by electron microscopy. A significant increase of HIV-1 p24 antigen was also detected in the culture supernatant. The syncytia formation was blocked by pretreating the transfectant with anti-human CD4 or by pretreating the MOLT-4/HIV with anti-HIV-1 serum obtained from an infected individual, indicating that the syncytia formed as a result of the interaction of human CD4 on the rabbit transfectant with the HIV-1 envelope protein expressed on MOLT-4/HIV. In contrast, only a very small proportion of the rabbit transfectants expressed HIV-1-specific antigens upon infection with an HIV-1 stock. This may indicate that, although rabbit cells have partially acquired susceptibility to HIV-1 by transfection of human CD4 gene, rabbit cells may further require such a molecule as might be provided by MOLT-4 to become fully susceptible to HIV-1 infection. The possibility of the rabbit as a model for HIV-1 infection is also discussed.     

Increased number of single-LTR HIV-1 DNA junctions correlates with HIV-1 antigen expression and CD4+ cell decline in vivo

Human immunodeficiency type 1 (HIV-1) DNA in peripheral blood cells of HIV-1 infected individuals may be present as integrated and/or unintegrated DNA. Several reports have indicated that a major proportion of HIV-1 DNA in the asymptomatic phase is linear, full-length, and unintegrated and in the symptomatic phase either circular unintegrated or integrated in the host genome. We developed a quantitative polymerase chain reaction (PCR) technique to detect single-LTR HIV-1 DNA junctions, reflecting the presence of unintegrated single-LTR circles. In vitro infection of a CD4+ T-cell line resulted first in the increase of single-LTR junctions followed by syncytium formation and a rise of p24 antigen production. The number of single-LTR HIV-1 DNA junctions was further studied in two acutely infected individuals and in 21 long-term infected individuals. The number of single-LTR junctions was significantly correlated with CD4+ cell decline, p24 antigen expression, and total HIV-1 DNA content of peripheral blood mononuclear cells (PBMC). Single-LTR HIV-1 DNA junctions were absent from PBMC containing other forms of HIV-1 DNA in four of nine non/slow progressors relative to 2 of 12 rapid progressors/AIDS patients. We conclude from our data that quantitative detection of single-LTR HIV-1 DNA junctions can be used as an early DNA marker of the transition from clinical latency to active replication in the peripheral blood. © 1995 Wiley-Liss, inc.     

HIV-1 Nef protein inhibits the in vitro induction of a specific antibody response to Candida albicans by an early up-regulation of IL-15 production

We have previously demonstrated that exogenous Nef protein induced activation of normal human T cells up-regulating IL-15 production by monocytes. Since HIV-1 infection results in the early impairment of immune functions we decided to evaluate if Nef is able to modulate the induction of a specific antibody response. Human peripheral blood mononuclear cells from healthy donors were induced in vitro to mount a specific antibody response to the Candida albicans antigen. We show that Nef inhibited, in a dose-dependent manner, the induction of the anti-C. albicans antibody response. The ability of an anti-Nef antibody to prevent such inhibition indicates that the effect was indeed Nef-specific. In the Nef-treated cultures an early increase of IL-15 production was observed and the addition of anti-IL-15 antibody abrogated the Nef-induced inhibitory effect. Moreover the addition of IL-15 to the cultures inhibited, as well as Nef, the induction of the specific antibody response. Thus, our results suggest that Nef may inhibit the induction of a specific antibody response by an early up-regulation of IL-15 production. A better comprehension of this phenomenon may be important for unravelling some aspects of the B cell defects in HIV infection.     

Intrathecal IgG synthesis and specificity of oligoclonal IgG in patients infected with HIV-1 do not correlate with CNS disease.

The CSF/serum immune response to HIV 1 was studied in 24 patients admitted for investigation. The level of antibody to HIV-1 and specificity of oligoclonal IgG were determined in blood and cerebrospinal fluid (CSF). The majority of patients demonstrated elevated levels of intrathecal IgG synthesis, with levels of HIV-1-specific antibody frequently being significantly higher in CSF than in serum. In 16 of 21 patients the CSF/serum antibody ratio indicated active intrathecal synthesis. Oligoclonal banding was present in CSF from all 24 patients. Immunoprinting of serum and CSF demonstrated antigenic specificity (p24, gp 160, RT) of the clonal antibodies in all of 12 patients though the patterns of reactivity in CSF did not necessarily correspond with that of serum. Although a specific association of particular patterns with HIV CNS disease was not found we feel that these markers should be included in longitudinal studies of HIV-related diseases of the CNS. The specificity of oligoclonal antibodies, both in CSF and in serum was demonstrated, and this specificity may be a useful marker for longitudinal studies in HIV-1 antibody-positive asymptomatic patients.     

Primary proliferative T cell response to wild-type p53 protein in patients with breast cancer

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. These are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half-life than wild-type p53 and accumulate in the nucleus of tumor cells. Anti-p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild-type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti-p53 vaccine development in tumor immunology.     

A longitudinal study of the relationship between galactosylation degree of IgG and rheumatoid factor titer and avidity during long-term immunization of rabbits with BSA

Although IgG with reduced content of galactose has been implicated as important in the autoimmune rheumatoid factor (RF) response in rheumatoid arthritis (RA), relatively little is known about the temporal relationship between RF and the degree of galactosylation of IgG in vivo. We established an experimental model for studying the dynamic association between changes in the relative extent of galactosylation of IgG antigen(s) and the main parameters of RF activity, such as the titer, specificity and functional affinity/avidity. Rabbits hyperimmunized with BSA were used for examining the influence of long-term antigenic stimulation on the galactosylation status of IgG and rheumatoid factor production. The results showed that the galactosylation profile of IgG varied during the humoral anti-BSA response in rabbits and that the accompanying RF response fluctuated in titer and binding avidity for differently galactosylated IgG. The immune complexes (IC) were found to be composed of differently galactosylated IgG differing in capacity to inhibit the agglutination activity of RF. Moreover, the ability of circulating RF to react avidly with rather small IC was associated with a lower content of galactose in complexed IgG. The results suggest that a certain dynamic relationship exists between the oligosaccharide moiety of IgG and the titer and avidity of RF during the normal anti-BSA response of rabbits.